Fig 1.
Cut and integration sites on the Act4 gene.
Homology arms of the constructs were designed to match the DSB ends generated by in vitro transcribed sgRNAs 64 (orange), 190 (red), and/or 234 (purple). sgRNA-145 (green), which was not co-injected but encoded in construct 64+234-perfect is also shown. Activities of all sgRNAs have been validated (Table F in S1 File). Arrows represent primers used to confirm integration site. Figure is not drawn to scale.
Fig 2.
Minimum integration rates and types of integration events as determined by fluorescence and PCR, respectively, for all injected constructs.
(A) Bar chart showing minimum integration rates which are calculated as follows: number of positive pools/total number of G0 survivors X 100%. Constructs with perfect homology arms are represented by open bars while those with recoded homology arms by filled boxes. P = plasmid. (B) Pie chart showing the proportion of canonical (black) to off-target/non-canonical (grey) integration events for each construct and their corresponding donor types. *N/A = Not applicable. PCR for these constructs were carried out on either pooled samples (n>5) or only one individual G1 from each pool (Table E in S1 File) rather than each individual positive G1.
Table 1.
Isolines established from G1 individuals and scoring of flight ability of trans-heterozygous females to test for allelism to AeAct4hdr1.
Table 2.
Concentration of each component in injection mixes.
Fig 3.
Characteristics of gene conversion tracts in perfect repair events generated with constructs 190-recoded (red bar) and 234-recoded (purple bar).
Positions of SNPs within the homology arms, relative to the cut sites for 190-recoded and 234-recoded, are marked with red and purple vertical lines, respectively (See Tables C and D in S1 File for specific nucleotide changes introduced). Count of HDR events indicates the number of individuals, and independent pools from which a conversion of that size range was recovered. Error bars indicate the range of possible conversion tract lengths which could not be detected by sequencing.
Fig 4.
Proposed mechanisms of synthesis-dependent strand annealing (SDSA) and mismatch repair.
(A) Double-stranded break caused by Cas9 is followed by 5’–3’ resection of the broken ends, leaving 3’ overhangs which will search for homology to initiate repair. (B) One of the overhanging strands finds homology in the donor and invades very closely to the homology arm-transgene junction, thereby bypassing the recoding on this side of the homology arms. The invading strand synthesises DNA using donor sequences as a template. (C) When this newly synthesised strand recognises homology on the 3’ overhang of the other end of the DSB, it dissociates from the template, anneals to the 3’ overhang, and is ligated to the 5’ end of one of the previously resected strands (squared in blue). (D) The other strand now repairs its break by DNA synthesis and ligation using the invading strand as its template, forming a heteroduplex region (squared in red). (E) Finally, the heteroduplex region is resolved by the mismatch repair pathway which favours the invading strand over the non-invading strand.